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Inhibitory effect of miRNA expression vector targeting GRP78 on proliferation of human esophageal carcinoma EC109 cells
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Abstract:
Objective:To explore the effect of miRNA eukaryotic expression vector targeting glucose regulated protein 78 ( GRP78 ) gene on proliferation of human esophageal carcinoma EC109 cells. Methods: Four pairs of specific miRNA interference sequences targeting GRP78 gene and one pair of negative control sequence were designed and synthesized. The recombined plasmids of miRNA targeting GRP78 , pcDNATM6.2-miR78-1, pcDNATM6.2-miR78-2, pcDNATM6.2-miR78-3 and pcDNATM6.2-miR78-4, were constructed using pcDNATM6.2-GW/EmGFP-miR eukaryotic expression vector, and were transfected into HEK293 cells by lipofectamine. After the treatment with blasticidin for two weeks, the stably transfected HEK293 cells were obtained. The transfection efficiency was observed by fluorescence microscopy. The best interference plasmid was then selected and transfected into EC109 cells. The expression of GRP78 mRNA and the proliferation of EC109 cells were detected by RT-PCR and CCK-8 assay, respectively. Results: Sequencing results indicated that four recombined plasmids of miRNA targeting GRP78 were successfully constructed. After transfection and screening, the expression of GFP was observed in all transfected HEK293 cells. The expression of GRP78 mRNA in HEK293 cells transfected with all the four interference plasmids was decreased (P<0.05), comparing with the untransfected or negative control group (pcDNATM6.2-Ctrl), in which the highest interference efficiency was observed in pcDNATM6.2-miR78-1 group. After pcDNATM6.2-miR78-1 transfection, the expression of GRP78 mRNA in EC109 cells was significantly decreased compared with the untransfected group and the pcDNATM6.2-Ctrl group (\[0.38±0.02\] vs \[1.03±004\], \[1.00±0.03\], P<0.05), respectively. CCK8 assay showed that EC109 cell proliferation was significantly suppressed after pcDNATM62-miR78-1 transfection in 12, 24, 48, 72 h (\[0.028±0.001\] vs \[0.086±0.010\], \[0.035±0.003\] vs \[0.155±0011\], \[0.112±0.009\] vs \[0.389±0.008\], \[0.169±0.013\] vs \[0.433±0.009\]; P<005). Conclusion: Four pairs of miRNA expression vectors targeting GRP78 gene are successfully constructed, in which pcDNATM62-miR78-1 shows the best gene silencing efficiency and efficiently inhibits the proliferation of EC109 cells.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 30972925)
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